Is a Glucose-6-Phosphate Dehydrogenase (G6PD) assay sufficient to diagnose G6PD deficiency?

Is G6PD Assay Sufficient to Diagnose G6PD Deficiency?

A G6PD assay is generally sufficient for diagnosing G6PD deficiency in most patients, but the type of assay matters significantly—qualitative tests work well for males and homozygous females with severe deficiency, while quantitative assays or flow cytometry are necessary for heterozygous females and patients with concurrent hematological conditions. 1

Assay Selection Based on Patient Population

Males and Homozygous Females

• Qualitative fluorescent spot tests are appropriate for detecting severe G6PD deficiency (enzyme activity <10% of normal) in males and homozygous females. 1
• The spectrophotometric assay reliably identifies deficient subjects in these populations. 2

Heterozygous Females

• Qualitative screening alone is insufficient for heterozygous females due to X-chromosome inactivation creating mosaic populations of normal and deficient red blood cells. 3
• Flow cytometry allows quantification of the percentage of G6PD-deficient RBCs, which varies among heterozygous females and provides more accurate diagnosis than spectrophotometry alone. 3
• Flow cytometry detected several cases of G6PD deficiency that were missed by spectrophotometry, especially in heterozygous females with normal or subnormal enzyme activity. 3

Critical Timing Considerations

When NOT to Test

• Avoid testing during or immediately after acute hemolytic episodes, as reticulocytes and young RBCs may contain near-normal enzyme levels, potentially masking deficiency. 4
• Wait at least 50 days after RBC transfusion before performing enzyme assays, as donor RBC contamination of 6-12% can cause false-negative results. 4
• Testing 120 days post-transfusion is ideal but often impractical in frequently transfused patients. 4

Patients with Anemia or Reticulocytosis

• The spectrophotometric assay is less reliable when other blood conditions are present, particularly anemia with reticulocytosis. 2
• Flow cytometry is less influenced by hemoglobin concentration, number of RBCs, and reticulocyte count compared to spectrophotometry. 2
• Consider calculating the PK/G6PD ratio or comparing to controls with similar reticulocyte counts when interpreting borderline results. 4

When Molecular Testing Is Needed

• Molecular analysis may be required in females heterozygous for the disorder when enzymatic testing is inconclusive. 5
• Genetic testing helps identify specific variants (Mediterranean vs. African), which determines hemolytic risk severity and drug contraindications. 1, 6
• The Mediterranean variant (Gdmed) causes life-threatening hemolysis requiring strict oxidant drug avoidance, while the African variant (GdA-) produces milder, self-limited hemolysis. 1, 6

Clinical Context for Testing

Pre-Treatment Screening

• Qualitative screening is recommended before starting oxidant drugs (dapsone, primaquine, sulfonamides) in patients of Mediterranean, African, Indian, or Southeast Asian descent. 1
• For P. vivax radical cure with tafenoquine, quantitative G6PD testing is required—do not use if activity is <70%. 1
• Primaquine, rasburicase, and methylene blue are absolutely contraindicated in G6PD deficiency. 1, 6

Patients with Intermediate Deficiency

• Quantitative testing is needed to determine the degree of deficiency in borderline cases, particularly when considering weekly primaquine dosing. 6
• Patients with 30-70% activity and non-Mediterranean variants can receive modified primaquine regimens with close monitoring. 1

Common Pitfalls

• False negatives occur in sickle cell disease patients even with flow cytometry. 3
• Neutropenia may be present but is not a reliable screening marker for G6PD deficiency. 1
• Testing whole blood without removing white cells and platelets prevents detection of deficiency in all cases examined. 4
• Buffy coat removal or filtration is essential for accurate RBC enzyme measurement. 4